This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have recently published our observation of the rapid effects of the lead Flex-Het compound, SHetA2, on mitochondrial density, membrane integrity, Cytochrome C release and ROS generation, all indicative of the intrinsic apoptosis pathway. More recently, we have observed that Flex-Hets are potent, uncompetitive inhibitors of mitochondrial electron transport chain complex I and also inhibitors of complex II. Since few inhibitors of both complex I and II are know at present, in the first part of this proposal we will further examine the effect of Flex-Het drugs on succinate dehydrogenase and full ubiquinone dependent succinate:ubiquinone oxidoreductase activity. However mitochondria targeting drugs may be a two-edged sword, ROS themselves are highly toxic to cells and mitochondria can gradually accumUlate oxidative damage leading to mitochondrial collapse and cell death. In the second part of this proposal we propose to assess the possibility of mitochondrial injury in heart and liver cells following long-term Flex-Het treatment. Markers of impaired mitochondrial function such as decreased cellular ATP, decreased mitochondrial glutathione and decreased complex I activity will be measured in both cells, mitochondria and submitochondrial particles isolated from snap-frozen heart and liver tissue obtained from animals sacrificed following necropsy in an ongoing long-term colon cancer chemoprevention project evaluating ShetA2.